Hacd2 deficiency in mice leads to an early and lethal mitochondrial disease.

OBJECTIVE: Mitochondria fuel most animal cells with ATP, ensuring proper energetic metabolism of organs. Early and extensive mitochondrial dysfunction often leads to severe disorders through multiorgan failure. Hacd2 gene encodes an enzyme involved in very long chain fatty acid (C ≥ 18) synthesis, yet its roles in vivo remain poorly understood. Since mitochondria function relies on specific properties of their membranes conferred by a particular phospholipid composition, we investigated if Hacd2 gene participates to mitochondrial integrity. METHODS: We generated two mouse models, the first one leading to a partial knockdown of Hacd2 expression and the second one, to a complete knockout of Hacd2 expression. We performed an in-depth analysis of the associated phenotypes, from whole organism to molecular scale. RESULTS: Thanks to these models, we show that Hacd2 displays an early and broad expression, and that its deficiency in mice is lethal. Specifically, partial knockdown of Hacd2 expression leads to death within one to four weeks after birth, from a sudden growth arrest followed by cachexia and lethargy. The total knockout of Hacd2 is even more severe, characterized by embryonic lethality around E9.5 following developmental arrest and pronounced cardiovascular malformations. In-depth mechanistic analysis revealed that Hacd2 deficiency causes altered mitochondrial efficiency and ultrastructure, as well as accumulation of oxidized cardiolipin. CONCLUSIONS: Altogether, these data indicate that the Hacd2 gene is essential for energetic metabolism during embryonic and postnatal development, acting through the control of proper mitochondrial organization and function.

Cardiolipin content controls mitochondrial coupling and energetic efficiency in muscle.

Unbalanced energy partitioning participates in the rise of obesity, a major public health concern in many countries. Increasing basal energy expenditure has been proposed as a strategy to fight obesity yet raises efficiency and safety concerns. Here, we show that mice deficient for a muscle-specific enzyme of very-long-chain fatty acid synthesis display increased basal energy expenditure and protection against high-fat diet-induced obesity. Mechanistically, muscle-specific modulation of the very-long-chain fatty acid pathway was associated with a reduced content of the inner mitochondrial membrane phospholipid cardiolipin and a blunted coupling efficiency between the respiratory chain and adenosine 5'-triphosphate (ATP) synthase, which was restored by cardiolipin enrichment. Our study reveals that selective increase of lipid oxidative capacities in skeletal muscle, through the cardiolipin-dependent lowering of mitochondrial ATP production, provides an effective option against obesity at the whole-body level.

The Role of Cullin-RING Ligases in Striated Muscle Development, Function, and Disease.

The well-orchestrated turnover of proteins in cross-striated muscles is one of the fundamental processes required for muscle cell function and survival. Dysfunction of the intricate protein degradation machinery is often associated with development of cardiac and skeletal muscle myopathies. Most muscle proteins are degraded by the ubiquitin-proteasome system (UPS). The UPS involves a number of enzymes, including E3-ligases, which tightly control which protein substrates are marked for degradation by the proteasome. Recent data reveal that E3-ligases of the cullin family play more diverse and crucial roles in cross striated muscles than previously anticipated. This review highlights some of the findings on the multifaceted functions of cullin-RING E3-ligases, their substrate adapters, muscle protein substrates, and regulatory proteins, such as the Cop9 signalosome, for the development of cross striated muscles, and their roles in the etiology of myopathies.

Murine obscurin and Obsl1 have functionally redundant roles in sarcolemmal integrity, sarcoplasmic reticulum organization, and muscle metabolism.

Biological roles of obscurin and its close homolog Obsl1 (obscurin-like 1) have been enigmatic. While obscurin is highly expressed in striated muscles, Obsl1 is found ubiquitously. Accordingly, obscurin mutations have been linked to myopathies, whereas mutations in Obsl1 result in 3M-growth syndrome. To further study unique and redundant functions of these closely related proteins, we generated and characterized Obsl1 knockouts. Global Obsl1 knockouts are embryonically lethal. In contrast, skeletal muscle-specific Obsl1 knockouts show a benign phenotype similar to obscurin knockouts. Only deletion of both proteins and removal of their functional redundancy revealed their roles for sarcolemmal stability and sarcoplasmic reticulum organization. To gain unbiased insights into changes to the muscle proteome, we analyzed tibialis anterior and soleus muscles by mass spectrometry, uncovering additional changes to the muscle metabolism. Our analyses suggest that all obscurin protein family members play functions for muscle membrane systems.

Cullin-3 dependent deregulation of ACTN1 represents a new pathogenic mechanism in nemaline myopathy.

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.

Transplantation of wild-type mouse hematopoietic stem and progenitor cells ameliorates deficits in a mouse model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an incurable autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin due to an intronic GAA-repeat expansion in the FXN gene. We report the therapeutic efficacy of transplanting wild-type mouse hematopoietic stem and progenitor cells (HSPCs) into the YG8R mouse model of FRDA. In the HSPC-transplanted YG8R mice, development of muscle weakness and locomotor deficits was abrogated as was degeneration of large sensory neurons in the dorsal root ganglia (DRGs) and mitochondrial capacity was improved in brain, skeletal muscle, and heart. Transplanted HSPCs engrafted and then differentiated into microglia in the brain and spinal cord and into macrophages in the DRGs, heart, and muscle of YG8R FRDA mice. We observed the transfer of wild-type frataxin and Cox8 mitochondrial proteins from HSPC-derived microglia/macrophages to FRDA mouse neurons and muscle myocytes in vivo. Our results show the HSPC-mediated phenotypic rescue of FRDA in YG8R mice and suggest that this approach should be investigated further as a strategy for treating FRDA.

Targeted Lipidomic Analysis of Myoblasts by GC-MS and LC-MS/MS.

Lipids represent ∼10% of the cell dry mass and play essential roles in membrane composition and physical properties, energy storage, and signaling pathways. In the developing or the regenerating skeletal muscle, modifications in the content or the flipping between leaflets of membrane lipid components can modulate the fusion capacity of myoblasts, thus constituting one of the regulatory mechanisms underlying myofiber growth. Recently, few genes controlling these qualitative and quantitative modifications have started to be unraveled. The precise functional characterization of these genes requires both qualitative and quantitative evaluations of a global lipid profile. Here, we describe a lipidomic protocol using mass spectrometry, allowing assessing the content of fatty acids, glycerophospholipids, and cholesterol in the routinely used C2C12 mouse myoblast cell line, or in primary cultures of mouse myoblasts.

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation.

The role of cullin E3-ubiquitin ligases for muscle homeostasis is best known during muscle atrophy, as the cullin-1 substrate adaptor atrogin-1 is among the most well-characterized muscle atrogins. We investigated whether cullin activity was also crucial during terminal myoblast differentiation and aggregation of acetylcholine receptors for the establishment of neuromuscular junctions in vitro. The activity of cullin E3-ligases is modulated through post-translational modification with the small ubiquitin-like modifier nedd8. Using either the Nae1 inhibitor MLN4924 (Pevonedistat) or siRNA against nedd8 in early or late stages of differentiation on C2C12 myoblasts, and primary satellite cells from mouse and human, we show that cullin E3-ligase activity is necessary for each step of the muscle cell differentiation program in vitro. We further investigate known transcriptional repressors for terminal muscle differentiation, namely ZBTB38, Bhlhe41, and Id1. Due to their identified roles for terminal muscle differentiation, we hypothesize that the accumulation of these potential cullin E3-ligase substrates may be partially responsible for the observed phenotype. MLN4924 is currently undergoing clinical trials in cancer patients, and our experiments highlight concerns on the homeostasis and regenerative capacity of muscles in these patients who often experience cachexia.

Progressive Structural Defects in Canine Centronuclear Myopathy Indicate a Role for HACD1 in Maintaining Skeletal Muscle Membrane Systems.

Mutations in HACD1/PTPLA cause recessive congenital myopathies in humans and dogs. Hydroxyacyl-coA dehydratases are required for elongation of very long chain fatty acids, and HACD1 has a role in early myogenesis, but the functions of this striated muscle-specific enzyme in more differentiated skeletal muscle remain unknown. Canine HACD1 deficiency is histopathologically classified as a centronuclear myopathy (CNM). We investigated the hypothesis that muscle from HACD1-deficient dogs has membrane abnormalities in common with CNMs with different genetic causes. We found progressive changes in tubuloreticular and sarcolemmal membranes and mislocalized triads and mitochondria in skeletal muscle from animals deficient in HACD1. Furthermore, comparable membranous abnormalities in cultured HACD1-deficient myotubes provide additional evidence that these defects are a primary consequence of altered HACD1 expression. Our novel findings, including T-tubule dilatation and disorganization, associated with defects in this additional CNM-associated gene provide a definitive pathophysiologic link with these disorders, confirm that dogs deficient in HACD1 are relevant models, and strengthen the evidence for a unifying pathogenesis in CNMs via defective membrane trafficking and excitation-contraction coupling in muscle. These results build on previous work by determining further functional roles of HACD1 in muscle and provide new insight into the pathology and pathogenetic mechanisms of HACD1 CNM. Consequently, alterations in membrane properties associated with HACD1 mutations should be investigated in humans with related phenotypes.

MLP and CARP are linked to chronic PKCα signalling in dilated cardiomyopathy.

MLP (muscle LIM protein)-deficient mice count among the first mouse models for dilated cardiomyopathy (DCM), yet the exact role of MLP in cardiac signalling processes is still enigmatic. Elevated PKCα signalling activity is known to be an important contributor to heart failure. Here we show that MLP directly inhibits the activity of PKCα. In end-stage DCM, PKCα is concentrated at the intercalated disc of cardiomyocytes, where it is sequestered by the adaptor protein CARP in a multiprotein complex together with PLCß1. In mice deficient for both MLP and CARP the chronic PKCα signalling chain at the intercalated disc is broken and they remain healthy. Our results suggest that the main role of MLP in heart lies in the direct inhibition of PKCα and that chronic uninhibited PKCα activity at the intercalated disc in the absence of functional MLP leads to heart failure.

HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth.

The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥ C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism.

Semaphorin-PlexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1.

Sprouting angiogenesis expands the embryonic vasculature enabling survival and homeostasis. Yet how the angiogenic capacity to form sprouts is allocated among endothelial cells (ECs) to guarantee the reproducible anatomy of stereotypical vascular beds remains unclear. Here we show that Sema-PlxnD1 signaling, previously implicated in sprout guidance, represses angiogenic potential to ensure the proper abundance and stereotypical distribution of the trunk's segmental arteries (SeAs). We find that Sema-PlxnD1 signaling exerts this effect by antagonizing the proangiogenic activity of vascular endothelial growth factor (VEGF). Specifically, Sema-PlxnD1 signaling ensures the proper endothelial abundance of soluble flt1 (sflt1), an alternatively spliced form of the VEGF receptor Flt1 encoding a potent secreted decoy. Hence, Sema-PlxnD1 signaling regulates distinct but related aspects of angiogenesis: the spatial allocation of angiogenic capacity within a primary vessel and sprout guidance.